Benzoyl-coenzyme A:glycine N-acyltransferase and phenylacetyl-coenzyme A:glycine N-acyltransferase from bovine liver mitochondria. Purification and characterization.

Two closely related acyl-CoA:amino acid N-acyl-transferases were purified to near-homogeneity from preparations of bovine liver mitochondria. Each enzyme consisted of a single polypeptide chain with a molecular weight near 33,000. One transferase was specific for benzoyl-CoA, salicyl-CoA, and certain short straight and branched chain fatty acyl-CoA esters as substrates while the other enzyme specifically used either phenylacetyl-CoA or indoleacetyl-CoA. Acyl-CoA substrates for one transferase inhibited the other. Glycine was the preferred acyl acceptor for both enzymes but either L-asparagine or L-glutamine also served. Peptide products for each transferase were identified by mass spectrometry. Enzymatic cleavage of acyl-CoA was stoichiometric with release of thiol and formation of peptide product. Apparent Km values were low for the preferred acyl-CoA substrates relative to the amino acid acceptors (10(-5) M range compared to greater than 10(-3) M). Both enzymes were inhibited by high nonphysiological concentrations of certain divalent cations (Mg2+, Ni2+, and Zn2+). In contrast to benzoyltransferase, phenylacetyltransferase was sensitive to inhibition by either 10(-4) M 5,5'-dithiobis(2-nitrobenzoate) or 10(-5) M p-chloromercuribenzoate; 10(-4) M phenylacetyl-CoA partially protected phenylacetyltransferase against 5,5'-dithiobis(2-nitrobenzoate) inactivation but 10(-1) M glycine did not. For activity, phenylacetyltransferase required addition of certain monovalent cations (K+, Rb+, Na+, Li+, Cs+, or (NH4)+) to the assay system but benzoyltransferase did not. Preliminary kinetic studies of both transferases were consistent with a sequential reaction mechanism in which the acyl-CoA substrate adds to the enzyme first, glycine adds before CoA leaves, and the peptide product dissociates last.     

Acyl-CoA:glycine N-acyltransferase: organelle localization and affinity toward straight- and branched-chained acyl-CoA esters in rat liver

Prompted by the fact that the urinary excretion of organic acids in the riboflavin-deficient rat closely mimics that found in patients with inborn errors in the acyl-CoA dehydrogenation systems, the organelle localization and the apparent kinetic constants (Km and Vmax values) for the rat liver acyl-CoA:glycine-N-acyltransferase (glycine-N-acylase) toward isobutyryl-CoA, 2-methylbutyryl-CoA, isovaleryl-CoA, butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, and benzoyl-CoA were determined. The studies on organelle localization demonstrated that the glycine-N-acylase is exclusively an intramitochondrial enzyme, and that no activity is present in peroxisomes, which also possess ability to produce Acyl-CoAs. The kinetic studies were done in order to elucidate whether the quantitative differences in excretion profile of acylglycines between riboflavin-deficient rats and patients with beta-oxidation defects are caused by differences in ability to conjugate the various acyl-CoAs. It was found that the Km values for the rat liver enzyme were generally somewhat lower than the values found in man, but with the same chain length profile. Consequently, the above-mentioned differences in excretion profile of acylglycines between riboflavin-deficient rats and patients with beta-oxidation defects cannot be explained by differences in affinity toward the glycine-N-acylase.     

Reversible lysine acetylation regulates activity of human glycine N-acyltransferase-like 2 (hGLYATL2): implications for production of glycine-conjugated signaling molecules

Lysine acetylation is a major post-translational modification of proteins and regulates many physiological processes such as metabolism, cell migration, aging, and inflammation. Proteomic studies have identified numerous lysine-acetylated proteins in human and mouse models (Kim, S. C., Sprung, R., Chen, Y., Xu, Y., Ball, H., Pei, J., Cheng, T., Kho, Y., Xiao, H., Xiao, L., Grishin, N. V., White, M., Yang, X. J., and Zhao, Y. (2006) Mol. Cell 23, 607-618). One family of proteins identified in this study was the murine glycine N-acyltransferase (GLYAT) enzymes, which are acetylated on lysine 19. Lysine 19 is a conserved residue in human glycine N-acyltransferase-like 2 (hGLYATL2) and in several other species, showing that this residue may be important for enzyme function. Mutation of lysine 19 in recombinant hGLYATL2 to glutamine (K19Q) and arginine (K19R) resulted in a 50-80% lower production of N-oleoyl glycine and N-arachidonoylglycine, indicating that lysine 19 is important for enzyme function. LC/MS/MS confirmed that Lys-19 is not acetylated in wild-type hGLYATL2, indicating that Lys-19 requires to be deacetylated for full activity. The hGLYATL2 enzyme conjugates medium- and long-chain saturated and unsaturated acyl-CoA esters to glycine, resulting in the production of N-oleoyl glycine and also N-arachidonoyl glycine. N-Oleoyl glycine and N-arachidonoyl glycine are structurally and functionally related to endocannabinoids and have been identified as signaling molecules that regulate functions like the perception of pain and body temperature and also have anti-inflammatory properties. In conclusion, acetylation of lysine(s) in hGLYATL2 regulates the enzyme activity, thus linking post-translational modification of proteins with the production of biological signaling molecules, the N-acyl glycines.     

Effects of secondary interactions on the kinetics of peptide and peptide ester hydrolysis by tissue kallikrein and trypsin

Kinetic constants for the hydrolysis by porcine tissue beta-kallikrein B and by bovine trypsin of a number of peptides related to the sequence of kininogen (also one containing a P2 glycine residue instead of phenylalanine) and of a series of corresponding arginyl peptide esters with various apolar P2 residues have been determined under strictly comparative conditions. kcat and kcat/Km values for the hydrolysis of the Arg-Ser bonds of the peptides by trypsin are conspicuously high. kcat for the best of the peptide substrates, Ac-Phe-Arg-Ser-Val-NH2, even reaches kcat for the corresponding methyl ester, indicating rate-limiting deacylation also in the hydrolysis of a peptide bond by this enzyme. kcat/Km for the hydrolysis of the peptide esters with different nonpolar L-amino acids in P2 is remarkably constant (range 1.7), as it is for the pair of the above pentapeptides with P2 glycine or phenylalanine. kcat for the ester substrates varies fivefold, however, being greatest for the P2 glycine compounds. Obviously, an increased potential of a P2 residue for interactions with the enzyme lowers the rate of deacylation. In contrast to results obtained with chymotrypsin and pancreatic elastase, trypsin is well able to tolerate a P3 proline residue. In the hydrolysis of peptide esters, tissue kallikrein is definitely superior to trypsin. Conversely, peptide bonds are hydrolyzed less efficiently by tissue kallikrein and the acylation reaction is rate-limiting. The influence of the length of peptide substrates is similar in both enzymes and indicates an extension of the substrate recognition site from subsite S3 to at least S'3 of tissue kallikrein and the importance of a hydrogen bond between the P3 carbonyl group and Gly-216 of the enzymes. Tissue kallikrein also tolerates a P3 proline residue well. In sharp contrast to the behaviour of trypsin is the very strong influence of the P2 residue in tissue-kallikrein-catalyzed reactions. kcat/Km varies 75-fold in the series of the dipeptide esters with nonpolar L-amino acid residues in P2, a P2 glycine residue furnishing the worst and phenylalanine the best substrate, whereas this exchange in the pentapeptides changes kcat/Km as much as 730-fold. This behaviour, together with the high value of kcat/Km for Ac-Phe-Arg-OMe of 3.75 X 10(7) M-1 s-1, suggests rate-limiting binding (k1) in the hydrolysis of the best ester substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specificity of diacylglycerol acyltransferase from bovine mammary gland, liver and adipose tissue towards acyl-CoA esters.

1. Microsomal diacylglycerol acyltransferase from bovine lactating mammary gland, liver and adipose tissue was capable of acylating microsomal-bound 1,2-dipalmitolyglycerol with acyl-CoA of chain length C4--C18. 2. The activity of the liver and adipose enzymes towards butyryl-CoA and hexanoyl-CoA relative to longer-chain acyl-CoA was similar to that of the mammary enzyme. The Km and V values of the three enzymes with butyryl-CoA and hexanoyl-CoA were similar, except for the V values of the adipose enzyme which were higher. 3. Microsomal diacylglycerol acyltransferase from mammary gland and liver of non-ruminants was also capable of utilizing butyryl-CoA. 4. These results indicate that the unique presence of short-chain acids in ruminant milk triacylglycerols is not caused by differences in specificity between the diacylglycerol acyltransferase from ruminant mammary and other tissues.     

Nucleophilic addition of glycine esters to Gd@C82

The first investigation of nucleophilic addition of glycine esters to Gd@C₈₂ is reported and hydroxyl was found to compete with glycine esters in the reaction. The multiple adducts containing either mere glycine esters or both glycine esters and hydroxyl group were identified by MALDI TOF mass spectrometry and characterized with UV-Vis-NIR spectrometry. Because glycine ethyl ester is more soluble than glycine methyl ester in alcohol and toluene, as many as eight glycine ethyl ester groups can be added to the metallofullerene cage while the maximal number for glycine methyl ester groups is only four.     

Comparison of the kinetics and mechanism of the papain-catalyzed hydrolysis of esters and thiono esters

The kinetic constants for the papain-catalyzed hydrolysis of the methyl thiono esters of N-benzoylglycine and N-(beta-phenylpropionyl)glycine are compared with those for the corresponding methyl ester substrates. The k2/Ks values for the thiono esters are 2-3 times higher than those for the esters, and both show bell-shaped pH dependencies with similar pKa's (approximately 4 and 9). The k3 values for the thiono esters are 30-60 times less than those for the esters and do not exhibit a pH dependency. Solvent deuterium isotope effects on k2/Ks and k3 were measured for the ester and thiono ester substrates of both glycine derivatives. Each thiono ester substrate showed an isotope effect similar to that for the corresponding ester substrate. Moreover, use of the proton inventory technique indicated that, as for esters, one proton is transferred in the transition state for deacylation during reactions involving thiono esters and the degree of heavy atom reorganization in the transition state is very similar in both cases. The k3 values for the hydrolysis of a series of para-substituted N-benzoylglycine esters were found to correlate with the k3 values for the corresponding para-substituted thiono esters [Carey, P. R., Lee, H., Ozaki, Y., & Storer, A. C. (1984) J. Am. Chem. Soc. 106, 8258-8262], showing that the rate-determining step for the deacylation of both thiolacyl and dithioacyl enzymes probably involves the disruption of a contact between the substrate's glycinic nitrogen atom and the sulfur of cysteine-25. It is concluded that the hydrolysis of esters and thiono esters proceeds by essentially the same reaction pathway. Due to an oxygen-sulfur exchange process the product released in the case of the N-(beta-phenylpropionyl)glycine thiono ester substrate is the dioxygen acid; however, for the N-benzoylglycine thiono ester substrate, the thiol acid is the initial product. This thiol acid then acts as a substrate for papain and reacylates the enzyme to eventually give the dioxygen acid product. It is shown that thiol acids are excellent substrates for papain.     

Effects of riboflavin deficiency and clofibrate treatment on the five acyl-CoA dehydrogenases in rat liver mitochondria.

Rats were maintained on a riboflavin-deficient diet or on a diet containing clofibrate (0.5%, w/w). The activities of the mitochondrial FAD-dependent straight-chain acyl-CoA dehydrogenases (butyryl-CoA, octanoyl-CoA and palmitoyl-CoA) and the branched-chain acyl-CoA dehydrogenases (isovaleryl-CoA and isobutyryl-CoA) involved in the degradation of branched-chain acyl-CoA esters derived from branched-chain amino acids were assayed in liver mitochondrial extracts prepared in the absence and presence of exogenous FAD. These activities were low in livers from riboflavin-deficient rats (11, 28, 16, 6 and less than 2% of controls respectively) when prepared in the absence of exogenous FAD, and were not restored to control values when prepared in 25 microM-FAD (29, 47, 28, 7 and 17%). Clofibrate feeding increased the activities of butyryl-CoA, octanoyl-CoA and palmitoyl-CoA dehydrogenases (by 48, 116 and 98% of controls respectively), but not, by contrast, the activities of isovaleryl-CoA and isobutyryl-CoA dehydrogenases (62 and 102% of controls respectively). The mitochondrial fractions from riboflavin-deficient and from clofibrate-fed rats oxidized palmitoylcarnitine in State 3 at rates of 32 and 163% respectively of those from control rats.     

Peroxisomal beta-oxidation of long-chain fatty acids possessing different extents of unsaturation.

Rates of peroxisomal beta-oxidation were measured as fatty acyl-CoA-dependent NAD+ reduction, by using solubilized peroxisomal fractions isolated from livers of rats treated with clofibrate. Medium- to long-chain saturated fatty acyl-CoA esters as well as long-chain polyunsaturated fatty acyl-CoA esters were used. Peroxisomal beta-oxidation shows optimal specificity towards long-chain polyunsaturated acyl-CoA esters. Eicosa-8,11,14-trienoyl-CoA, eicosa-11,14,17-trienoyl-CoA and docosa-7,10,13,16-tetraenoyl-CoA all gave Vmax. values of about 150% of that obtained with palmitoyl-CoA. The Km values obtained with these fatty acyl-CoA esters were 17 +/- 6, 13 +/- 4 and 22 +/- 3 microM respectively, which are in the same range as the value for palmitoyl-CoA (13.8 +/- 1 microM). Myristoyl-CoA gave the higher Vmax. (110% of the palmitoyl-CoA value) of the saturated fatty acyl-CoAs tested. Substrate inhibition was mostly observed with acyl-CoA esters giving Vmax. values higher than 50% of that given by palmitoyl-CoA.     

Biosynthesis of triacylglycerols containing short-chain fatty acids in lactating cow mammary gland. Activity of diacylglycerol acyltransferase towards short-chain acyl-CoA esters

1. Microsomal 1,2-diacylglycerol acyltransferase from lactating cow mammary gland incorporated equal molar amounts of microsomal-bound 1,2-dipalmitoyl [2-3H]glycerol and [1-14C]-butyrate, [1-14C]hexanoate or [1-14C]palmitate from their CoA esters into triacylglycerol. The enzyme could also utilize exogenous 1,2-diacylglycerols in the presence of ethanol. 2. The pH optimum of the enzyme was 6.1 and 6.4 with butyryl-CoA and hexanoyl-CoA respectively. Values of V were approximately the same (2.7 and 2.4 nmol-min-1-mg-1, respectively), but values of Km were different (34 and 10 muM, respectively) with these two substrates. Mg2+ was not required as cofactor. 3. The presence ofa Mg2+-dependent phosphatidate phosphatase in the microsomal fraction was demonstrated. 4. It is proposed that triacylglycerols containing butyric and hexanoic acid are biosynthesized in cow mammary gland by the glycerolphosphate pathway, in which long-chain 1,2-diacylglycerols derived from phosphatidic acid are acylated at the sn-3 position by short-chain acyl-CoA esters.     

The complement component C1s catalysed hydrolysis of peptide 4-nitroanilide substrates

The kinetic parameter kcat/Km has been determined for the hydrolysis of peptide 4-nitroanilides, catalysed by complement component C1s. Substrates based on the C-terminal sequence of human C4a (Leu-Gln-Arg) were synthesised. Replacement of the glutamine residue by glycine or serine increased kcat/Km. Substitution of valine for the leucine residue increased kcat/Km, while substitution of glycine or lysine for the leucine residue decreased kcat/Km slightly. D-Val-Ser-Arg 4-nitroanilide is the most reactive 4-nitroanilide substrate towards C1s, so far. These results are discussed in relation to the amino acid sequences near the bonds cleaved by C1s in C4, C2 and C1 inhibitor.     

Selective inhibition of acyl-CoA dehydrogenases by a metabolite of hypoglycin.

Extracts of liver mitochondria from donor rats given hypoglycin, the toxic amino acid from the ackee plant (Blighia sapida) showed drastically reduced levels of acyl-CoA dehydrogenase activity with butyryl-CoA as substrate. Activity with octanoyl- and palmitoyl-CoA was unaffected. Evidence that the active agent is methylenecyclopropylacetyl-CoA, a hypoglycin metabolite, was obtained by observing effects of the compound on a partially purified enzyme mixture prepared from rabbit liver. At 13 muM concentration, it strongly inhibited butyryl-CoA dehydrogenase (EC 1.3.99.2) with butyryl-CoA as substrate; it was far less effective with palmitoyl-CoA as substrate for the other similar enzymes present in the preparation. Unlike normal substrates of the acyl-CoA dehydrogenases, the compound itself, and not a reaction product, is inhibitory. The observed effect is consistent with quite general inhibition of fatty acid beta-oxidation by hypoglycin.     

Comparison of the kinetics of the papain-catalyzed hydrolysis of glycine- and alanine-based esters and thiono esters

The kinetic constants for the papain-catalyzed hydrolysis of a series of substrates with glycine or alanine in the P1 position are discussed. The substrates have N-benzoyl, N-(p-nitrobenzoyl), N-(beta-phenylpropionyl), or N-(methyloxycarbonyl)phenylalanine attached to the P1 moiety, and kinetic constants are obtained for both esters and thiono esters. The results for the hydrolysis of esters can be readily interpreted in terms of the known specificity of papain. For any glycine ester the change in kcat/Km upon substituting C=S for C=O or upon substituting an alpha-CH3 group is minimal. However, upon making both these substitutions, i.e., going from a glycine ester to an alanine thiono ester substrate, larger changes are seen for this ratio. Data for N-benzoyl- and N-(beta-phenylpropionyl)glycine and -alanine methyl thiono esters show that k2 is the parameter most affected by the double C=S and alpha-CH3 substitution. A further conclusion is that the deacylation rate constants for any pair of glycine and alanine dithioacyl papains are similar; e.g., for the intermediates based on the "good" substrates PheAla and PheGly k3 differs by only 20%. This is a surprising finding in light of the very different conformations and interactions of the bound acyl groups revealed by resonance Raman spectroscopy and raises the possibility that specific stereochemical effects, such as the oxyanion hole and general base catalysis, are not operating in the hydrolysis of dithioacyl papains.     

Purification and properties of an acetoacetyl coenzyme A-reacting phosphotransbutyrylase from Clostridium beijerinckii ("Clostridium butylicum") NRRL B593

During the study of acetoacetyl coenzyme A (CoA)-reacting enzymes of Clostridium beijerinckii NRRL B593, a phosphate-dependent acetoacetyl-CoA-utilizing activity was detected in protein fractions devoid of thiolase and phosphotransacetylase. Further purification of this acetoacetyl-CoA-utilizing activity yielded an enzyme which may be designated as phosphotransbutyrylase (PTB; phosphate butyryltransferase [EC 2.3.1.19]). PTB from C. beijerinckii NRRL B593 was purified 160-fold with a yield of 14% and, with the best fractions, purified 190-fold to near homogeneity. It showed a native Mr of 205,000 and a subunit Mr of 33,000. PTB activity was sensitive to pH changes within the physiological range of 6 to 8. PTB exhibited a broad substrate specificity. The Km values at pH 7.5 for butyryl-CoA, acetoacetyl-CoA, and acetyl-CoA were 0.04, 1.10, and 3.33 mM, respectively. The Vmax values with butyryl-CoA and acetoacetyl-CoA were comparable, but the Vmax/Km was higher for butyryl-CoA than for acetoacetyl-CoA. An apparent Km of 6.5 mM for phosphate was obtained with butyryl-CoA as the cosubstrate, whereas it was 12.9 mM with acetoacetyl-CoA as the cosubstrate. It remains to be established whether the putative compound acetoacetyl phosphate is produced in the PTB-catalyzed reaction with acetoacetyl-CoA.     

Gender-related effects of chronic ethanol ingestion on rat hepatic acyl-CoA:cholesterol acyltransferase

The influence of chronic ethanol ingestion on hepatic acyl-CoA: cholesterol acyltransferase activity was investigated to determine the relationship between alcohol intake and cholesterol ester accumulation. Rats were given nutritionally complete liquid diets supplemented with 6.3% ethanol or an isocaloric equivalent of dextrin-maltose for 5 weeks. During this period, the hepatic acyl-CoA: cholesterol acyltransferase activity of ethanol-fed male rats remained constant, whereas the same activity in pair-fed controls as well as chow-fed rats exhibited a 30% decrease in activity. Unlike alcohol-fed male rats, the hepatic acyl-CoA: cholesterol acyltransferase activity of female rats decreased by approximately 30% by the fifth week of ethanol ingestion. Despite the fact that the gender of the animals led to disparate levels of acyl-CoA: cholesterol acyltransferase activity in response to ethanol ingestion, similar levels of cholesteryl ester accumulation were observed. The altered levels of acyl-CoA: cholesterol acyltransferase activity caused no significant change in the cholesterol concentration, cholesterol/phospholipid ratio, phospholipid fatty acid composition, or the membrane fluidity of the hepatic microsomes. We conclude that the altered hepatic acyl-CoA: cholesterol acyltransferase activity of ethanol-fed female rats cannot be directly responsible for ethanol-induced accumulation of cholesteryl esters.     

Monounsaturated fatty acyl-coenzyme A is predictive of atherosclerosis in human apoB-100 transgenic, LDLr-/- mice

ACAT2, the enzyme responsible for the formation of cholesteryl esters incorporated into apolipoprotein B-containing lipoproteins by the small intestine and liver, forms predominantly cholesteryl oleate from acyl-CoA and free cholesterol. The accumulation of cholesteryl oleate in plasma lipoproteins has been found to be predictive of atherosclerosis. Accordingly, a method was developed in which fatty acyl-CoA subspecies could be extracted from mouse liver and quantified. Analyses were performed on liver tissue from mice fed one of four diets enriched with one particular type of dietary fatty acid: saturated, monounsaturated, n-3 polyunsaturated, or n-6 polyunsaturated. We found that the hepatic fatty acyl-CoA pools reflected the fatty acid composition of the diet fed. The highest percentage of fatty acyl-CoAs across all diet groups was in monoacyl-CoAs, and values were 36% and 46% for the n-3 and n-6 polyunsaturated diet groups and 55% and 62% in the saturated and monounsaturated diet groups, respectively. The percentage of hepatic acyl-CoA as oleoyl-CoA was also highly correlated to liver cholesteryl ester, plasma cholesterol, LDL molecular weight, and atherosclerosis extent. These data suggest that replacing monounsaturated with polyunsaturated fat can benefit coronary heart disease by reducing the availability of oleoyl-CoA in the substrate pool of hepatic ACAT2, thereby reducing cholesteryl oleate secretion and accumulation in plasma lipoproteins.     

Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD.

The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtilis. We have identified, through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310-bp segment of the cloned region revealed a single gene (opuD) whose product (OpuD) was essential for glycine betaine uptake and osmoprotection in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity. An opuD mutant was constructed by marker replacement, and the OpuD-mediated glycine betaine uptake activity was compared with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glycine betaine uptake systems. These mutants were used to determine the kinetic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, with Km values in the low micromolar range, which should allow B. subtilis to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity, demonstrating that three transport systems for this osmoprotectant operate in B. subtilis.     

Pharmacological properties of glycine uptake in the developing rat retina

A pharmacological characterization of glycine transport was performed in the rat retina at different postnatal ages. The uptake of 3H-glycine increased during the first 2 weeks of postnatal age, reaching maximum values at 12 days; then it decreased sharply to the adult values. We found a Na+ -dependent and high-affinity transport system with a Km of 100 microM. The Na+ Hill coefficient for glycine uptake was 1.76 +/- 0.07. Although glycine uptake was insensitive to staurosporine and phorbol ester, it was reduced 40-50% by sarcosine and ALX5407. Besides, amoxapine inhibited glycine uptake by 40 and 70% in adult and immature retina, respectively. These results suggest that the Glyt1 transporter was concentrated in the nerve terminals. In addition to the presence of Glyt1 in the retina, our results provided evidence of the occurrence of Glyt2 and/or another isoform of glycine transporter, which might have had a role in the retina development.     

Acyl coenzyme A carboxylase of Propionibacterium shermanii: detection and properties.

An acyl coenzyme A (CoA) carboxylase, which catalyzes the adenosine triphosphate-dependent fixation of CO2 into acetyl-, propionyl-, and butyryl-CoA, was detected in fractionated cell extracts of Propionibacterium shermanii. Catalytic activity was inhibited by avidin but was unaffected by avidin pretreated with excess biotin. The carboxylase levels detected were relatively small and were related to cellular growth. Maximal carboxylase activity was detected in cells grown for about 96 h. Thereafter, the activity declined rapidly. Optimal CO2 fixation occurred at pH 7.5. Other parameters of the assay system were optimized, and the apparent Km values for substrates were determined. The end product of the reaction (with acetyl-CoA as the substrate) was identified as malonyl-CoA. The stoichiometry of the reaction was such that, for every mole of acetyl-CoA and adenosine triphosphate consumed, 1 mol each of malonyl-CoA, adenosine diphosphate, and orthophosphate was formed. These data provide the first evidence for the presence of another biotin-containing enzyme, an acyl-CoA carboxylase, in these bacteria in addition to the well-characterized methylmalonyl-CoA carboxyltransferase.     

Detection of acyl-coenzyme A thioester intermediates of fatty acid beta-oxidation as the N-acylglycines by negative-ion chemical ionization gas chromatography-mass spectrometry.

An analytical method for the separation and quantitation of acyl-CoA thioesters by gas chromatography-mass spectrometry is described. The method utilizes glycine aminolysis of the acyl-CoA thiolesters, esterification with pentafluorobenzyl bromide followed by gas chromatographic separation, and detection by negative chemical ionization mass spectrometry of the N-acylpentafluorobenzyl glycinates. The glycine aminolysis provides over 100-fold discrimination against oxygen esters and obviates the difficulty of removing trace contaminants of free fatty acids. The limit of detection of the described methodology for palmitoyl-CoA has been found to be 300 fmol, which improves at shorter chain lengths. Baseline separation was obtained for a standard mixture of seven acyl-CoAs (60 pmol injected) containing butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, decanoyl-CoA, lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA. The above procedure is also applicable to the alpha-beta unsaturated and 3-hydroxyacyl-CoA derivatives, making it possible to quantify all of the intermediates in fatty acid oxidation, except the 3-ketoacyl-CoAs, in a single procedure.